Novel megakaryocytic protein tyrosine kinase 1

ABSTRACT

The present invention relates to novel cytoplasmic tyrosine kinases isolated from megakaryocytes (megakaryocyte kinases or MKKs) which are involved in cellular signal transduction pathways and to the use of these novel proteins in the diagnosis and treatment of disease. The present invention further relates to specific megakaryocyte kinases, designated MKK1, MKK2 and MKK3, and their use as diagnostic and therapeutic agents.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a Division of U.S. application Ser. No. 09/977,260,filed Oct. 16, 2001, incorporated herein by reference in its entirety,which is a Division of U.S. application Ser. No. 08/232,545, filed Apr.22, 1994, incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Introduction

The present invention relates to novel cytoplasmic tyrosine kinasesisolated from megakaryocytes (megakaryocyte kinases or MKKs) which areinvolved in cellular signal transduction pathways and to the use ofthese novel proteins in the diagnosis and treatment of disease.

The present invention further relates to specific megakaryocyte kinases,designated MKK1, MKK2 and MKK3, and their use as diagnostic andtherapeutic agents.

2. Background

Cellular signal transduction is a fundamental mechanism whereby externalstimuli that regulate diverse cellular processes are relayed to theinterior of cells. These processes include, but are not limited to, cellproliferation, differentiation and survival. Many tyrosine kinases areexpressed in postmitotic, fully differentiated cells, particularly inthe case of hematopoietic cells, and it seems likely that these proteinsare involved in specialized cellular functions that are specific for thecell types in which they are expressed. (Eiseman, E. and J. B. Bolen,Cancer cells 2(10):303-310, 1990). A central feature of signaltransduction is the reversible phosphorylation of certain proteins (forreviews, see Posada, J. and Cooper, J. A., 1992, Mol. Biol. Cell3:583-392; Hardie, D. G., 1990, Symp. Soc. Exp. Biol. 44:241-255). Thephosphorylation state of a protein is modified through the reciprocalactions of tyrosine kinases (TKs), which function tophosphorylateproteins, and tyrosine phosphatases (TPs), which function todephosphorylate proteins. Normal cellular function requires a delicatebalance between the activities of these two types of enzyme.

Phosphorylation of cell surface tyrosine kinases, stimulates a physicalassociation of the activated receptor with intracellular targetmolecules. Some of the target molecules are in turn phosphorylated.Other target molecules are not phosphorylated, but assist in signaltransmission by acting as adapter molecules for secondary signaltransducer proteins.

The secondary signal transducer molecules generated by activatedreceptors result in a signal cascade that regulates cell functions suchas cell division or differentiation. Reviews describing intracellularsignal transduction include Aaronson, S. A., Science 254:1146-1153,1991; Schlessinger, J. Trends Biochem. Sci. 13:443-447, 1988; andUllrich, A., and Schlessinger, J. Cell 61:203-212, 1990.

Receptor tyrosine kinases are composed of at least three domains: anextracellular ligand binding domain, a transmembrane domain and acytoplasmiccatalytic domain that can phosphorylate tyrosine residues.The intracellular, cytoplasmic, nonreceptor protein tyrosine kinases maybe broadly defined as those protein tyrosine kinases which do notcontain a hydrophobic, transmembrane domain. Bolen (Oncogene, vol. 8,pgs. 2025-2031 (1993)) reports that 24 individual protein tyrosinekinases comprising eight different families of non-receptor proteintyrosine kinases have been identified: Ab1/Arg; Jak1/Jak2/Tyk2; Fak;Fes/Fps; Syk/Zap; Tsk/Tec/Atk; Csk; and the Src group, which includesthe family members Src, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk. Allof the non-receptor protein tyrosine kinases are thought to be involvedin signaling pathways that modulate growth and differentiation. Bolen,supra, suggests that half of the nonreceptor protein tyrosine kinaseshave demonstrated oncogenic potential and half appear to be primarilyrelated to suppressing the activity of Src-related protein kinases andcould be classified as anti-oncogenes.

While distinct in their overall molecular structure, each member of agiven morphotypic family of cytoplasmic protein tyrosine kinases sharessequence homology in certain non-catalytic domains in addition tosharing sequence homology in the catalytic kinase domain. Examples ofdefined non-catalytic domains include the SH2 (SRC homology domain 2;Sadowski, I et al., Mol. Cell. Biol. 6:4396-4408; Kock, C. A. et al.,1991, Science 252:668-674) domains, SH3 domains (Mayer, B. J. et al.,1988, Nature 332:269272) and PH domains (Musacchio et al., TIBS18:343-348 (1993). These non-catalytic domains are thought to beimportant in the regulation of protein-protein interactions duringsignal transduction (Pawson, T. and Gish, G., 1992, Cell 71:359-362).

While the metabolic roles of cytoplasmic protein tyrosine kinases areless well understood than that of the receptor-type protein tyrosinekinases, significant progress has been made in elucidating some or theprocesses in which this class of molecules is involved. For example,members of the src family, lck and fyn, have been shown to interact withCD4/CD8 and the T cell receptor complex, and are thus implicated in Tcell activation, (Veillette, A. Davidson, D., 1992, TIG 8:61-66). Somecytoplasmic protein tyrosine kinases have been linked to certain phasesof the cell cycle (Morgan, D. O. et al., 1989, Cell 57:775-786; Kipreos,E. T. et al., 1990, Science 248:217-220; Weaver et al., 1991, Mol. Cell.Biol. 11:4415-4422), and cytoplasmic protein tyrosine kinases have beenimplicated in neuronal and hematopoietic development (Maness, P., 1992,Dev. Neurosci 14:257-270 and Rawlings et al., Science 261:358-361(1993)). Deregulation of kinase activity through mutation oroverexpression is a well-established mechanism underlying celltransformation (Hunter et al., 1965, supra; Ullrich et al., supra).

A variety of cytoplasmic tyrosine kinases are expressed in, and may haveimportant functions in, hematopoietic cells including src, lyn, fyn,blk, lck, csk and hck. (Eisenian, E. and J. B. Bolen, Cancer Cells2(10):303-310, 1990). T-cell activation, for example, is associated withactivation of lck. The signaling activity of lyn may be stimulated bybinding of allergens to IgE on the surface of basophils. (Eisenian,supra).

Abnormalities in tyrosine kinase regulated signal transduction pathwayscan result in a number of disease states. For example, mutations in thecytoplasmic tyrosine kinase atk (also called btk) are responsible forthe x-linked agammaglobulinemia, (Ventrie, D., et al., Nature361:226-23, 1993). This defect appears to prevent the normaldifferentiation of pre-B cells to mature circulating B cells and resultsin a complete lack of serum immunoglobulins of all isotypes. Thecytoplasmic tyrosine kinase Zap-70 has been suggested as indispensablefor the development of CD8 single-positive T cells as well as for signaltransduction and function of single positive CD4 T cells, and lack ofthis protein leads of an immunodeficiency disease in humans, (Arpala,E., et al., Cell 76:1-20, 1994). Gene knockout experiments in micesuggest a role for src in the regulation of osteociast function and boneremodeling as these mice develop osteopetrosis. (Soriano et al., Cell64:693-702, 1991 and Lowe et al., PNAS (in press)).

Megakaryocytes are large cells normally present in bone marrow andspleen and are the progenitor cell for blood platelets. Megakaryocytesare associated with such disease states as acute megakaryocytic leukemia(Lu et al., Cancer Genet Cvtoaenet, 67(2):81-89 (1993) and Moody et al.,Pediatr Radiol. 19(6-7):486-488 (1989)), a disease that is difficult todiagnose early and which is characterized by aberrant proliferation ofimmature cells or “blasts;” myelofibrosis (Smith et al., Crit Rev OncolHematol. 10(4 :305-314 (1990) and Marino, J. Am. Osteopath Assoc.10:1323-1326 (1989)), an often fatal disease where the malignant cellmay be of megakaryocytic lineage and may be mediated by platelet ormegakaryocyte growth factors; acute megakaryocytic myelosis (Fohlmeisteret al., Haematologia 19(2):151-160 (1986)) a rapidly fatal diseasecharacterized by megakaryocytic proliferation and the appearance ofimmature megakaryocytes in the circulation; and acute myelosclerosis(Butler et al., Cancer 49(12):2497-2499 (1982) and Bearman et al.,Cancer 43(1):279-93 (1979)) a myeloproliferative syndrome where themarrow is characterized by atypical megakaryocytes.

Platelets play a key role in the regulation of blood clotting and woundhealing, as well as being associated with such disease conditions asthrombocytopenia, atherosclerosis, restenosis and leukemia. Severalreceptor tyrosine kinases have been identified in human megakaryocytesincluding c-kit, blg and blk. (Hoffman, H., Blood 74:1196-1212, 1989;Long, M. W., Stem Cells 11:33-40, 1993; Zaebo, K. M., et al., Cell63:213-224,1990). Cytoplasmic tyrosine kinases of human megakaryocyticorigin have also been reported. (Bennett et al., Journal of BiologicalChemistry 289(2):1068-1074, 1994; Lee et al., Gene 1-5, 1993; and Sakanoet al., Oncogene 9:1155-1161 (1994)).

SUMMARY OF THE INVENTION

The present invention relates to novel, cytoplasmic tyrosine kinasesisolated from megakaryocytes (megakaryocyte kinases or MKKs) which areinvolved in cellular signal transduction pathways. Particular MKKsdescribed herein are referred to as MKK1, MKK2, and MKK3. The completenucleotide sequences encoding MKK1, MKK2, and MKK3 are disclosed herein,and provide the basis for several aspects of the invention hereinafterdescribed.

The present invention is based, in part, upon the discovery that MKK1,MKK2, and MKK3 have amino acid and structural homology, respectively, tothe PTKs csk (Brauninger et al. Gene, 110:205-211 (1992) and Brauningeret al., Oncoaene, 8:1365-1369 (1993)), atk/btk, tec and tsk (Vetrie etal., Nature 361:226-233 (1993); Mano et al., Oncogene 8:417-424 (1993)and Heyeck et al., PNAS USA 90:669-673, 1993, respectively) and fyn(Kawakami et al. Mol. Cell. Bio 6:4195-4201, 1986)).

The present invention also relates, in part, to nucleotide sequences andexpression vectors encoding MKKs. Also described herein are methods oftreatment and diagnosis of diseases resulting from abnormalities insignal transduction pathways in which MKKs are involved.

The MKK sequences disclosed herein may be used to detect and quantifylevels of MKK mRNA in cells and furthermore for diagnostic purposes fordetection of expression of MKKs in cells. For example, an MKK sequencemay be used in hybridization assays of biopsied tissue to diagnoseabnormalities in gene expression associated with a transformedphenotype.

Also disclosed herein are methods of treatment of diseases or conditionsassociated with abnormalities in signal transduction pathways inmegakaryocytes. Such abnormalities can result in, for example, underproduction of mature, differentiated cells, inappropriate proliferationof immature cells or modulation of activity of other important cellularfunctions.

Anti-MKK antibodies may be used for diagnostic purposes for thedetection of MKKs in tissues and cells. Anti-MKK antibodies may also beused for therapeutic purposes, for example, in neutralizing the activityof an MKK associated with a signal transduction pathway.

Oligonucleotide sequences, including anti-sense RNA and DNA moleculesand ribozymes, designed to inhibit the translation of MKK mRNA, may beused therapeutically in the treatment of disease states associated withaberrant expression of MKKs. In a particular embodiment of the inventiondescribed by way of Example 9 herein, an anti-MKK1 antisense molecule isused to inhibit MKK-1 protein synthesis resulting in reducedmegakaryocyte growth and differentiation.

Proteins, peptides and organic molecules capable of modulating activityof MKKs may be used therapeutically in the treatment of disease statesassociated with aberrant expression of MKKs. Alternatively, proteins,peptides and organic molecules capable of modulating activity of MKKsmaybe used therapeutically to enhance normal activity levels of MKKs.For example, small molecules found to stimulate MKK1 activity inmegakaryocytes may be used for ex vivo culturing of megakaryocytesintended for autologous treatment of patients receiving chemotherapy orother therapies which deplete megakaryoctyes or platelets, or in thetreatment of thrombocytopenia.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B. Human MKK1 nucleotide sequence (SEQ ID NO:1) anddeduced amino acid sequence (SEQ ID NO:2). Marked regions show thesignal sequence, the SH2 and SH3 domains, and the catalytic domain.

FIGS. 2A and 2B. Human MKK2 nucleotide sequence (SEQ ID NO:3) anddeduced amino acid sequence (SEQ ID NO:4). Marked regions show thesignal sequence, the pleckstrin homology domain (PH), the proline richsequences following the PH domain, the SH2 and SH3 domains, and thecatalytic domain.

FIGS. 3A and 3B. Human MKK3 nucleotide sequence (SEQ ID NO:5) anddeduced amino acid sequence (SEQ ID NO:6). Marked regions show thesignal sequence, the SH2 and SH3 domains, and the catalytic domain.

FIG. 4. Expression of MKK1 and MKK2 in human and rodent cell lines.

FIG. 5. Immunoprecipitation (i.p.) of in vitro transcribed andtranslated MKK1 and MKK2 proteins. Samples in lanes designated 1 through9 are as follows: 1. MKK1 i.p. with anti-carboxy terminus MKK1 Ab, 2.and 3. MKK1 i.p. with anti-amino terminus MKK1 Ab, 4. MKK1 i.p. withrabbit pre immune sera, 5. MKK2 i.p. with rabbit pre immune sera, 6,and, 7. MKK2 i.p. with anti-carboxy terminus MKK2 Ab, 8. MKK1 in vitrotranscribed/translated protein without i.p., 9. MKK2 in vitrotranscribed/translated protein without i.p.

FIGS. 6A and 6B. FIGS. 6A-6B illustrate anti-sense MMK1 expressionsuppresses AChE Production in primary murine bone marrow cultures. FIG.6A illustrates AChE production. FIG. 6B illustrates MKK1 proteinexpression.

FIG. 7. MKK2 and MKK3 autophosphorylate and transphosphorylate proteinswhen expressed in bacteria. Lanes 2, 4, and 6 represent non-inducedbacteria expressing MKK1, MKK2, MKK3, respectively. Lanes 1, 3, and 5represent induced bacteria expressing MKK1, MKK2, MKK3, respectively.

FIG. 8. MKK expression constructs.

FIG. 9. Shared amino acid sequence homology of MKK1 SEQ ID NO: 2 and cskSEQ ID NO: 7.

FIGS. 10A and 10B SEQ ID NOS 4, 8-10, respectively, in order ofappearance. Shared amino acid sequence homology of MKK2 and atk/btk.

FIGS. 11A, 11B, 11C and 11D SEQ ID NOS 6, 11-19, respectively, in orderof appearance. Shared amino acid sequence homology of MKK3 and srctyrosine kinase family members.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to novel, cytosolic megakaryocytic kinasesreferred to herein as “MKKs,” and in particular to megakaryocyte kinase1 (MKK1), megakaryocyte kinase 2 (MKK2), which are expressed in humanmegakaryocytic cell lines, and megakaryocyte kinase 3 (MKK3).

As used herein, MKK is a term which refers to MKK1, MKK2 and MKK3 fromany species, including, bovine, ovine, porcine, equine, murine andpreferably human, in naturally occurring-sequence or in variant form, orfrom any source, whether natural, synthetic, or recombinant. A preferredMKK variant is one having at least 80% amino acid homology, aparticularly preferred MKK variant is one having at least 90% sequencehomology and another particularly preferred MKK variant is one having atleast 95% amino acid homology to the naturally occurring MKK.

MKK1 is a cytosolic tyrosine kinase of molecular weight 58 kD, asdetermined by SDS gel electrophoresis, having homology to the TK csk(Partanen, et al., Oncogene 6:2013-2018 (1991) and Nada et al., Nature351:69-72 (1991)) in the intervening sequences of its catalytic domain,the SH2 and SH3 domains, and other non-catalytic regions and like csk,lacks regulatory phosphorylation sites corresponding to c-src tyrosines416 and 527. MKK1 also lacks an amino-terminal myristylation site.

Csk is a recently described novel cytoplasmic TK that seems to play akey role in regulation of signal transduction in hematopoietic andneural development. For example csk has been shown to negativelyregulate members of the src family of TKs, including c-src, lck, andfyn, through its ability to phosphorylate regulatory tyrosines. (Bergmanet al., The EMBO Journal 11(8)8:2919-2924 (1992) and Sabe et al.,Molecular and Cellular Biology 12(10):4706-4713 (1992)). Autero et al.,(Molecular and Cellular Biology 14(2):1308-1321 (1994)) have reportedthat csk positively regulates a phosphatase, CD45, that is key to T-cellactivity. Csk mediated phosphorylation of CD45 phosphotyrosinephosphatase (PTPase) caused a several fold increase in its PTPaseactivity. Csk appears to play a role as a regulator of the sequence ofboth phosphorylation and dephosphorylation events culminating in cellactivation and proliferation.

Defective expression of csk in mouse embryos results in defects in theneural tube with subsequent death between day 9 and day 10 of gestation,with cells derived from these embryos exhibiting an order of magnitudeincrease in activity of src kinase (Nada et al., Cell 73:1125-1135(1993)). Overexpression of csk in transformed rat 3Y1 fibroblasts wasshown to cause reversion to normal phenotypes (Sabe et al., Molecularand Cellular Biology 12:4706-4713 (1992)).

MKK1 has 54% homology with csk at the amino acid level and structuralsimilarity to csk, i.e., the lack of regulatory phosphorylation sitesand the lack of an amino-terminal myristylation site. Experimental data,see Section 9, show that expression of human antisense MKK 1 sequencesinhibits synthesis of murine MKK 1, which inhibition is associated witha reduction of proliferation of megakaryocytes in vitro. Based upon theexperimental data in Section 9 and amino acid and structural homologywith csk, MKK1 appears to play a regulatory role in the growth anddifferentiation of megakaryocytes and perhaps neural tissues based onits expression in those tissues.

MKK2 is a novel cytosolic tyrosine kinase of molecular weight 78 kD, asdetermined by SDS gel electrophoresis, having homology to the tecsubfamily of TKs which also incudes tsk and atk/btk. Like the tecsubfamily, MKK2 lacks an amino-terminal site for myristylation and has aputative pleckstrin homology binding domain located 5′ to the SH3 domain(Musacchio et al., TIBS 18:343-348 (1993)). The pleckstrin homology (PH)domain has been found in a number of proteins with diverse cellularfunctions and is abundant in proteins involved in signal transductionpathways. Musacchio et al., supra suggest that the PH domain may beinvolved in molecular recognition similarly to SH2 and SH3 domains.

The tec family of tyrosine kinases appear to play roles in cellulardifferentiation and include family members tec, a kinase which may bespecifically involved in the cell growth of hepatocytes orhepatocarcinogenesis (Mano et al., supra); tsk, which may play a role inearly T-lymphocyte differentiation (Heyek et al., PNAS USA 90:669-673(1993)) and atk/btk. Aberrant expression of atk/btk has been shown to beresponsible for X-linked agammaglobulinemia (XLA), a human diseaseresulting from a developmental block in the transition from preB cellsto mature B cells (Ventrie, D. et al., supra).

MKK2 has 50% homology to atkibtk at the amino acid level and structuralsimilarity to tec family members, i.e., the presence of the SH2, SH3 andPH domains and the lack of an amino-terminal site for myristylation andthe carboxyl site of tyrosine phosphorylation found in family members.Based upon the amino acid homology and structural similarity to tecfamily members which play roles in cellular differentiation, MKK2 mayplay a role in the differentiation of megakaryoctyes.

MKK3 is a novel cytosolic tyrosine kinase of molecular weight 58 kD, asdetermined by SDS gel electrophoresis, having homology to the TK fyn.MKK3 does not have a myristylation sites. MKK3 does have a putativeregulatory cite at tyr 387 but the surrounding 12 amino acids are notidentical with other members of the src subfamily that share highlyconserved sequences in this region. MKK3 has 47% homology with fyn atthe amino acid level.

The fyn gene was originally characterized in normal human fibroblast andendothelial cells, but it is also expressed in a variety of other celltypes. Alternative splicing of fyn has been shown to yield two distincttranscripts, both coding for enzymatically active forms of the kinases.

MKK sequences could be used diagnostically to measure expression of MKKsin disease states, such as for example leukemia, where abnormalproliferation of immature myeloid cells occurs, or where abnormaldifferentiation of megakaryocytes occurs. MKKs could also be usedtherapeutically in the treatment of disease states involving abnormalproliferation or differentiation through interruption of signaltransduction by modulation of protein tyrosine kinases.

The nucleotide and deduced amino acid sequence of human MKK 1, MKK2, andMKK3 are shown in FIGS. 1A-1B (SEQ ID NOS 1-2), 2A-2B (SEQ ID NOS 3-4)and 3A-3B (SEQ ID NOS 5-6), respectively. FIGS. 9 (SEQ ID NOS 2 and 7,respectively, in order of appearance), 10A-10B (SEQ ID NOS 4, 8-10,respectively, in order of appearance) and 11A-11D (SEQ ID NOS 6, 11-19,respectively, in order of appearance) show the shared sequence homologybetween MKKs and related tyrosine kinases.

The MKK Coding Sequences

The nucleotide coding sequence and deduced amino acid sequence of thehuman MKK1, MKK2, and MKK3 genes are depicted in FIGS. 1A-1B (SEQ ID NOS1-2), 2A-2B (SEQ ID NOS 3-4) and 3A-3B (SEQ ID NOS 5-6), respectively.In accordance with the invention, any nucleotide sequence which encodesthe amino acid sequence of an MKK gene product can be used to generaterecombinant molecules which direct the expression of an MKK.

In a specific embodiment described herein, the human MKK1, MKK2, andMKK3 genes were isolated by performing polymerase chain reactions (PCR)in combination with two degenerate oligonucleotide primer pools thatwere designed on the basis of highly conserved sequences within thekinase domain of receptor tyrosine kinases corresponding to the aminoacid sequence HRDLAA (residues 350-355 of SEQ ID NO: 2) (sense primer)and SDVWSF/Y (SEQ ID NO: 24) (antisense primer) (Hanks et al., 1988).The MKK cDNAs were synthesized by reverse transcription of poly-A RNAfrom the human K-562 cell line, ATCC accession number CCL 243, or fromthe Meg 01 cell line, (Ogura et al., Blood 66:1384 (1985)).

The PCR fragments were used to screen a lambda gt11 library of humanfetal brain. For each individual MKK, several overlapping clones wereidentified. The composite of the cDNA clones for MKK1, MKK2, and MKK3are depicted in FIGS. 1A-1B (SEQ ID NOS 1-2), 2A-2B (SEQ ID NOS 3-4),and 3A-3B (SEQ ID NOS 5-6), respectively.

Further characterization of the individual MKKs is found infra.

Expression of MKK

In accordance with the invention, MKK polynucleotide sequences whichencode MKKs, peptide fragments of MKKs, MKK fusion proteins orfunctional equivalents thereof, may be used to generate recombinant DNAmolecules that direct the expression of MKK protein, MKK peptidefragment, fusion proteins or a functional equivalent thereof, inappropriate host cells. Such MKK polynucleotide sequences, as well asother polynucleotides which selectively hybridize to at least a part ofsuch MKK polynucleotides or their complements, may also be used innucleic acid hybridization assays, Southern and Northern blot analyses,etc.

Due to the inherent degeneracy of the genetic code, other DNA sequenceswhich encode substantially the same or a functionally equivalent aminoacid sequence, may be used in the practice of the invention for thecloning and expression of the MKK protein. Such DNA sequences includethose which are capable of hybridizing to the human MKK sequence understringent conditions. The phrase “stringent conditions” as used hereinrefers to those hybridizing conditions that (1) employ low ionicstrength and high temperature for washing, for example, 0.015 MNaC1/0.0015 M sodium citrate/0.1% SDS at 50° C.; (2) employ duringhybridization a denaturing agent such as formamide, for example, 50%(vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMNaCl, 75 mM sodium citrate at 42° C.; or (3) employ 50% formamide, 5×SSC(0.75 M NaCl, 0.075 M Sodium pyrophosphate, 5× Denhardt's solution,sonicated salmon sperm DNA (50 g/ml), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2×SSC and 0.1% SDS.

Altered DNA sequences which may be used in accordance with the inventioninclude deletions, additions or substitutions of different nucleotideresidues resulting in a sequence that encodes the same or a functionallyequivalent gene product. The gene product itself may contain deletions,additions or substitutions of amino acid residues within an MKKsequence, which result in a silent change thus producing a functionallyequivalent MKK. Such amino acid substitutions may be made on the basisof similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipatic nature of the residues involved.For example, negatively charged amino acids include aspartic acid andglutamic acid; positively charged amino acids include lysine andarginine; amino acids with uncharged polar head groups having similarhydrophilicity values include the following: leucine, isoleucine,valine; glycine, alanine; asparagine, glutamine; serine, threonine;phenylalanine, tyrosine.

The DNA sequences of the invention may be engineered in order to alteran MKK coding sequence for a variety of ends including but not limitedto alterations which modify processing and expression of the geneproduct. For example, mutations may be introduced using techniques whichare well known in the art, e.g., site-directed mutagenesis, to insertnew restriction sites, to alter glycosylation patterns, phosphorylation,etc.

In another embodiment of the invention, an MKK or a modified MKKsequence may be ligated to a heterologous sequence to encode a fusionprotein. For example, for screening of peptide libraries for inhibitorsof MKK activity, it may be useful to encode a chimeric MKK proteinexpressing a heterologous epitope that is recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between an MKK sequence and the heterologousprotein sequence, so that the MKK may be cleaved away from theheterologous moiety.

In an alternate embodiment of the invention, the coding sequence of anMKK could be synthesized in whole or in part, using chemical methodswell known in the art. See, for example, Caruthers et al., 1980, Nuc.Acids Res. Symp. Ser. 7:215-233; Crea and Horn, 180, Nuc. Acids Res.9(10):2331; Matteucci and Caruthers, 1980, Tetrahedron Letters 21:719;and Chow and Kempe, 1981, Nuc. Acids Res. 9(12):2807-2817.Alternatively, the protein itself could be produced using chemicalmethods to synthesize an MKK amino acid sequence in whole or in part.For example, peptides can be synthesized by solid phase techniques,cleaved from the resin, and purified by preparative high performanceliquid chromatography. (e.g., see Creighton, 1983, Proteins StructuresAnd Molecular Principles, W. H. Freeman and Co., N.Y. pp. 50-60). Thecomposition of the synthetic peptides may be confirmed by amino acidanalysis or sequencing (e.g., the Edman degradation procedure; seeCreighton, 1983, Proteins Structures and Molecular Principles, W. H.Freeman and Co., N.Y., pp. 34-49.

In order to express a biologically active MKK, the nucleotide sequencecoding for MKK, or a functional equivalent, is inserted into anappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence. The MKK gene products as well as host cells or celllines transfected or transformed with recombinant MKK expression vectorscan be used for a variety of purposes. These include but are not limitedto generating antibodies (i.e., monoclonal or polyclonal)thatcompetitively inhibit activity of an MKK and neutralize its activity.Anti-MKK antibodies may be used in detecting and quantifying expressionof an MKK in cells and tissues.

Expression Systems

Methods which are well known to those skilled in the art can be used toconstruct expression vectors containing an MKK coding sequence andappropriate transcriptional/translational control signals. These methodsinclude in vitro recombinant DNA techniques, synthetic techniques and invivo recombination/genetic recombination. See, for example, thetechniques described in Maniatis et al., 1989, Molecular Cloning ALaboratory Manual, Cold Spring Harbor Laboratory, N.Y. and Ausubel etal., 1989, Current Protocols in Molecular Biology, Greene PublishingAssociates and Wiley Interscience, N.Y.

A variety of host-expression vector systems may be utilized to expressan MKK coding sequence. These include but are not limited tomicroorganisms such as bacteria transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining an MKK coding sequence; yeast transformed with recombinantyeast expression vectors containing an MKK coding sequence; insect cellsystems infected with recombinant virus expression vectors (e.g.,baculovirus) containing an MKK coding sequence; plant cell systemsinfected with recombinant virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed withrecombinant plasmid expression vectors (e.g., Ti plasmid) containing anMKK coding sequence; or animal cell systems. The expression elements ofthese systems vary in their strength and specificities. Depending on thehost/vector system utilized, any of a number of suitable transcriptionand translation elements, including constitutive and induciblepromoters, may be used in the expression vector. For example, whencloning in bacterial systems, inducible promoters such as pL ofbacteriophage λ, plac, ptrp, ptac (ptrp-lac hybrid promoter) and thelike may be used; when cloning in insect cell systems, promoters such asthe baculovirus polyhedrin promoter may be used; when cloning in plantcell systems, promoters derived from the genome of plant cells (e.g.,heat shock promoters; the promoter for the small subunit of RUBISCO; thepromoter for the chlorophyll a/b binding protein) or from plant viruses(e.g., the 35S RNA promoter of CaMV; the coat protein promoter of TMV)may be used; when cloning in mammalian cell systems, promoters derivedfrom the genome of mammalian cells (e.g., metallothionein promoter) orfrom mammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5 K promoter) may be used; when generating cell lines thatcontain multiple copies of an MKK DNA, SV40-, BPV- and EBV-based vectorsmay be used with an appropriate selectable marker.

In bacterial systems a number of expression vectors may beadvantageously selected depending upon the use intended for the MKKexpressed. For example, when large quantities of MKK1 are to be producedfor the generation of antibodies, vectors which direct the expression ofhigh levels of fusion protein products that are readily purified may bedesirable. Such vectors include but are not limited to the E. coliexpression vector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in whichthe MKK1 coding sequence may be ligated into the vector in frame withthe lac Z coding region so that a hybrid AS-lac Z protein is produced;pIN vectors (Inouye & Inouye, 1985, Nucleic acids Res. 13:3101-3109; VanHeeke & Schuster, 1989, J. Biol. Chem. 264:5503-5509); and the like.pGEX vectors may also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. The pGEX vectors are designed to includethrombin or factor Xa protease cleavage sites so that the clonedpolypeptide of interest can be released from the GST moiety.

In yeast, a number of vectors containing constitutive or induciblepromoters may be used. For a review see, Current Protocols in MolecularBiology, Vol. 2, 1988, Ed. Ausubel et al., Greene Publish. Assoc. &Wiley Interscience, Ch. 13; Grant et al., 1987, Expression and SecretionVectors for Yeast, in Methods in Enzymology, Ed. Wu & Grossman, 1987,Acad. Press, N.Y. 153:516-544; Glover, 1986, DNA Cloning, Vol. II, IRLPress, Wash., D.C., Ch. 3; and Bitter, 1987, Heterologous GeneExpression in Yeast, Methods in Enzymology, Eds. Berger & Kimmel, Acad.Press, N.Y. 152:673-684; and The Molecular Biology of the YeastSaccharomyces, 1982, Eds. Strathern et al., Cold Spring Harbor Press,Vols. I and II.

In cases where plant expression vectors are used, the expression of anMKK coding sequence may be driven by any of a number of promoters. Forexample, viral promoters such as the 35S RNA and 19S RNA promoters ofCaMV (Brinson et al., 1984, Nature 310:511-514), or the coat proteinpromoter of TMV (Takamatsu et al., 1987, EMBO J. 6:307-311) may be used;alternatively, plant promoters such as the small subunit of RUBISCO(Coruzzi et al., 1984, EMBO J. 3:1671-1680; Broglie et al., 1984,Science 224:838-843); or heat shock promoters, e.g., soybean hsp 17.5-Eor hsp 17.3-B (Gurley et al., 1986, Mol. Cell. Biol. 6:559-565) may beused. These constructs can be introduced into plant cells using Tiplasmids, Ri plasmids, plant virus vectors, direct DNA transformation,microinjection, electroporation, etc. For reviews of such techniquessee, for example, Weissbach & Weissbach, 1988, Methods for PlantMolecular Biology, Academic Press, NY, Section VIII, pp. 421-463; andGrierson & Corey, 1988, Plant Molecular Biology, 2d Ed., Blackie,London, ch. 7-9.

An alternative expression system which could be used to express an MKKis an insect system. In one such system, Autographa californica nuclearpolyhidrosis virus (AcNPV) is used as a vector to express foreign genes.The virus grows in Spodoptera frugiperda cells. An MKK coding sequencemay be cloned into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample, the polyhedrin promoter). Successful insertion of an MKK codingsequence will result in inactivation of the polyhedrin gene andproduction of non-occluded recombinant virus (i.e., virus lacking theproteinaceous coat coded for by the polyhedrin gene). These recombinantviruses are then used to infect Spodoptera frugiperda cells in which theinserted gene is expressed. (e.g., see Smith et al., 1983, J. Viol.46:584; Smith, U.S. Pat. No. 4,215,051).

In mammalian host cells, a number of viral based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, an MKK coding sequence may be ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. This chimeric gene may then be inserted inthe adenovirus genome by in vitro or in vivo recombination. Insertion ina non-essential region of the viral genome (e.g., region E1 or E3) willresult in a recombinant virus that is viable and capable of expressingan MKK in infected hosts. (e.g., See Logan & Shenk, 1984, Proc. Natl.Acad. Sci. (USA) 81:3655-3659). Alternatively, the vaccinia 7.5 Kpromoter may be used. (See, e.g., Mackett et al., 1982, Proc. Natl.Acad. Sci. (USA) 79:7415-7419; Mackett et al., 1984, J. Virol.49:857-864; Panicali et al., 1982, Proc. Natl. Acad. Sci. 79:4927-4931).

Specific initiation signals may also be required for efficienttranslation of an inserted MKK coding sequences. These signals includethe ATG initiation codon and adjacent sequences. In cases where anentire MKK gene, including its own initiation codon and adjacentsequences, is inserted into the appropriate expression vector, noadditional translational control signals may be needed. However, incaseswhere only a portion of an MKK coding sequence is inserted, exogenoustranslational control signals, including the ATG initiation codon, mustbe provided. Furthermore, the initiation codon must be in phase with thereading frame of an MKK coding sequence to ensure translation of theentire insert. These exogenous translational control signals andinitiation codons can be of a variety of origins, both natural andsynthetic. The efficiency of expression may be enhanced by the inclusionof appropriate transcription enhancer elements, transcriptionterminators, etc. (see Bittner et al., 1987, Methods in Enzymol.153.516-544).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins. Appropriate cells lines or hostsystems can be chosen to ensure the correct modification and processingof the foreign protein expressed. To this end, eukaryotic host cellswhich possess the cellular machinery for proper processing of theprimary transcript, glycosylation, and phosphorylation of the geneproduct may be used. Such mammalian host cells include but are notlimited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, WI38, etc.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably express anMKK may be engineered. Rather than using expression vectors whichcontain viral origins of replication, host cells can be transformed withMKK DNA controlled by appropriate expression control elements (e.g.,promoter, enhancer, sequences, transcription terminators,polyadenylation sites, etc.), and a selectable marker. Following theintroduction of foreign DNA, engineered cells may be allowed to grow for1-2 days in an enriched media, and then are switched to a selectivemedia. The selectable marker in the recombinant plasmid confersresistance to the selection and allows cells to stably integrate theplasmid into their chromosomes and grow to form foci which in turn canbe cloned and expanded into cell lines. This method may advantageouslybe used to engineer cell lines which express an MKK.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adeninephosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection fordhfr, which confers resistance to methotrexate (Wigler et al., 1980,Natl. Acad. Sci. USA 77:3567; O'Hare et al., 1981, Natl. Acad. Sci. USA78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan &Berg, 1981), Natl. Acad. Sci. USA 78:2072); neo, which confersresistance to the aminoglycoside G-418 (Colberre-Garapin et al., 1981,J. Mol. Biol. 150:1); and hygro, which confers resistance to hygromycin(Santerre et al., 1984, Gene 30:147). Recently, additional selectablegenes have been described, namely trpB, which allows cells to utilizeindole in place of tryptophan; hisD, which allows cells to utilizehistinol in place of histidine (Hartman & Mulligan, 1988, Proc. Natl.Acad. Sci. USA 85:8047); and ODC (ornithine decarboxylase) which confersresistance to the omithine decarboxylase inhibitor,2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue L., 1987, In: CurrentCommunications in Molecular Biology, Cold Spring Harbor Laboratory,Ed.).

Identification of Transfectants or Transformants that Express the MKK

The host cells which contain the coding sequence and which express thebiologically active gene product may be identified by at least fourgeneral approaches; (a) DNA-DNA or DNA-RNA hybridization; (b) thepresence or absence of “marker” gene functions; (c) assessing the levelof transcription as measured by the expression of MKK mRNA transcriptsin the host cell; and (d) detection of the gene product as measured byimmunoassay or by its biological activity.

In the first approach, the presence of the MKK coding sequence insertedin the expression vector can be detected by DNA-DNA or DNA-RNAhybridization using probes comprising nucleotide sequences thatarehomologous to the MKK coding sequence, respectively, or portions orderivatives thereof.

In the second approach, the recombinant expression vector/host systemcan be identified and selected based upon the presence or absence ofcertain “marker” gene functions (e.g., thymidine kinase activity,resistance to antibiotics, resistance to methotrexate, transformationphenotype, occlusion body formation in baculovirus, etc.). For example,if the MKK1 coding sequence is inserted within a marker gene sequence ofthe vector, recombinant cells containing the MKK1 coding sequence can beidentified by the absence of the marker gene function. Alternatively, amarker gene can be placed in tandem with an MKK sequence under thecontrol of the same or different promoter used to control the expressionof the MKK coding sequence. Expression of the marker in response toinduction or selection indicates expression of the MKK coding sequence.

In the third approach, transcriptional activity for an MKK coding regioncan be assessed by hybridization assays. For example, RNA can beisolated and analyzed by Northern blot using a probe homologous to anMKK coding sequence or particular portions thereof. Alternatively, totalnucleic acids of the host cell may be extracted and assayed forhybridization to such probes.

In the fourth approach, the expression of an MKK protein product can beassessed immunologically, for example by Western blots, immunoassayssuch as radioimmuno-precipitation, enzyme-linked immunoassays and thelike.

Uses of MKK and Engineered Cell Lines

Megakaryocytes, the progenitor cell for blood platelets, and plateletsare associated with disease states involving aberrant proliferation ordifferentiation of such cells, such as acute megakaryocytic leukemia,acute megakaryocytic myelosis and thrombocytopenia. MKKs appear to playa role in the growth and differentiation of megkaryocytes, thereforeinhibitors of MKKs may be used therapeutically for the treatment ofdiseases states resulting from aberrant growth of megakaryocytes orplatelets. Alternatively, enhancers of MKKs may be used therapeuticallyto stimulate the proliferation of megakaryocytes in such applicationsas, for example, ex vivo culturing of megakaryocytes intended forautologous cell therapy in individuals receiving chemotherapy or othertherapies which deplete megakaryocytes or platelets or in treatingthrombocytopenia caused by other conditions.

In an embodiment of the invention, an MKK and/or cell line thatexpresses an MKK may be used to screen for antibodies, peptides, orother molecules that act as agonists or antagonists of MKK throughmodulation of signal transduction pathways. For example, anti-MKKantibodies capable of neutralizing the activity of MKK may be used toinhibit an MKK associated signal transduction pathway. Such antibodiescan act intracellularly utilizing the techniques described in Marasco etal. (PNAS 90:7889-7893 (1993) for example or through delivery byliposomes. Alternatively, screening of organic or peptide libraries withrecombinantly expressed MKK protein or cell lines expressing MKK proteinmay be useful for identification of therapeutic molecules that functionby modulating the kinase activity of MKK or its associated signaltransduction pathway. A therapeutic molecule may find application in adisease state associated with megakaryocytes, such as acutemegakaryocytic leukemia, or alternatively, in nondisease applications,for example in ex vivo culturing of megakaryocytes intended forautologous treatment of individuals undergoing chemotherapy. Syntheticcompounds, natural products, and other sources of potentiallybiologically active materials can be screened in a number of ways deemedto be routine to those of skill in the art.

The ability of antibodies, peptides, or other molecules to prevent ormimic, the effect of MKK on signal transduction responses on MKKexpressing cells may be measured. For example, responses such asactivation or inhibition of MKK kinase activity or modulation of secondmessenger production may be monitored. The term “second messenger” asused herein refers to any component or product found in the cascade ofsignal transduction events. These assays may be performed usingconventional techniques developed for these purposes.

Antibody Production and Screening

Various procedures known in the art may be used for the production ofantibodies to epitopes of the recombinantly produced MKK. Suchantibodies include but are not limited to polyclonal, monoclonal,chimeric, single chain, Fab fragments and fragments produced by a Fabexpression library. Neutralizing antibodies, i.e., those which inhibitthe biological activity, i.e., the kinase activity, of an MY areespecially preferred for diagnostics and therapeutics.

For the production of antibodies, various host animals may be immunizedby injection with an MKK protein including but not limited to rabbits,mice, rats, etc. Various adjuvants may be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacilli Calmette-Guerin) and Corynebacterium parvum.

Monoclonal antibodies to an MKK may be prepared by using any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include but are not limited to thehybridoma technique originally described by Koehler and Milstein,(Nature, 1975, 256:495-497), the human B-cell hybridoma technique(Kosbor et al., 1983, Immunology Today, 4:72; Cote et al., 1983, Proc.Natl. Acad. Sci, 80:2026-2030) and the EBV-hybridoma technique (Cole etal., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.,pp. 77-96). In addition, techniques developed for the production of“chimeric antibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci.81:6851-6855; Neuberger et al, 1984, Nature, 312:604-608; Takeda et al.,1985, Nature 314:452-454) by splicing the genes from a mouse antibodymolecule of appropriate antigen specificity together with genes from ahuman antibody molecule of appropriate biological activity can be used.Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778) can be adapted to produce anMKK-specific single chain antibodies.

Antibody fragments which contain specific binding sites of an MKK may begenerated by known techniques. For example, such fragments include butare not limited to: the F(ab′)₂ fragments which can be produced bypepsin digestion of the antibody molecule and the Fab fragments whichcan be generated by reducing the disulfide bridges of the F(ab′)₂fragments. Alternatively, Fab expression libraries may be constructed(Huse et al., 1989, Science 246:1275-1281) to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificitythe MKK of interest.

Screening of Peptide Library with MKK or MKK Engineered Cell Lines

Random peptide libraries consisting of all possible combinations ofamino acids attached to a solid phase support may be used to identifypeptides that are able to bind to MKK binding sites, e.g., SH2, SH3 orPH binding sites, or other functional domains of an MKK, such as kinasedomains. The screening of peptide libraries may have therapeutic valuein the discovery of pharmaceutical agents that act to stimulate orinhibit the biological activity of an MKK.

Identification of molecules that are able to bind to an MKK may beaccomplished by screening a peptide library with recombinant MKKprotein. Methods for expression of an MKK are described in Section 5.2,5.3 and 5.4 and may be used to express a recombinant full length MKK orfragments of an MKK depending on the functional domains of interest. Forexample, the kinase and SH2, SH3 or PH binding domains of an MKK may beseparately expressed and used to screen peptide libraries.

To identify and isolate the peptide/solid phase support that interactsand forms a complex with an MKK, it is necessary to label or “tag” theMKK molecule. The MKK protein may be conjugated to enzymes such asalkaline phosphatase or horseradish peroxidase or to other reagents suchas fluorescent labels which may include fluorescein isothyiocynate(FITC), phycoerythrin (PE) or rhodamine. Conjugation of any given labelto MKK may be performed using techniques that are routine in the art.Alternatively, MKK expression vectors may be engineered to express achimeric MKK protein containing an epitope for which a commerciallyavailable antibody exists. The epitope specific antibody may be taggedusing methods well known in the art including labeling with enzymes,fluorescent dyes or colored or magnetic beads.

The “tagged” MKK conjugate is incubated with the random peptide libraryfor 30 minutes to one hour at 22° C. to allow complex formation betweenan MKK and peptide species within the library. The library is thenwashed to remove any unbound MKK protein. If MKK has been conjugated toalkaline phosphatase or horseradish peroxidase the whole library ispoured into a petri dish containing a substrates for either alkalinephosphatase or peroxidase, for example, 5-bromo-4-chloro-3-indoylphosphate (BCIP) or 3,3′,4,4″-diamnobenzidine (DAB), respectively. Afterincubating for several minutes, the peptide/solid phase-MKK complexchanges color, and can be easily identified and isolated physicallyunder a dissecting microscope with a micromanipulator. If a fluorescenttagged MKK molecule has been used, complexes may be isolated byfluorescent activated sorting. If a chimeric MKK protein expressing aheterologous epitope has been used, detection of the peptide/MKK complexmay be accomplished by using a labeled epitope specific antibody. Onceisolated, the identity of the peptide attached to the solid phasesupport may be determined by peptide sequencing.

Screening of Organic Compounds with MKK Protein or Engineered Cell Lines

Cell lines that express an MKK may be used to screen for molecules thatmodulate MKK activity or signal transduction. Such molecules may includesmall organic or inorganic compounds or extracts of biological materialssuch as plants, fungi, etc., or other molecules that modulate MKKactivity or that promote or prevent MKK mediated signal transduction.Synthetic compounds, natural products, and other sources of potentiallybiologically active materials can be screened in a number of ways.

The ability of a test molecule to interfere with MKK signal transductionmay be measured using standard biochemical techniques. Other responsessuch as activation or suppression of catalytic activity, phosphorylationor dephosphorylation of other proteins, activation or modulation ofsecond messenger production, changes in cellular ion levels,association, dissociation or translocation of signalling molecules, ortranscription or translation of specific genes may also be monitored.These assays may be performed using conventional techniques developedfor these purposes in the course of screening. (See, for example,Peralidi, et al., J. Biochem. 285:71-78 (1992) or Campbell et al., JBC268:7427-7434 (1993)).

Cellular processes under the control of an MKK signalling pathway mayinclude, but are not limited to, normal cellular functions such asproliferation or differentiation of megakaryocytes or platelets, inaddition to abnormal or potentially deleterious processes such asunregulated or inappropriate cellproliferation, blocking ofdifferentiation of megakaryocytes or platelets, or ultimately celldeath. The qualitative or quantitative observation and measurement ofany of the described cellular processes by techniques known in the artmay be advantageously used as a means of scoring for signal transductionin the course of screening.

MKK, or functional derivatives thereof, useful in identifying compoundscapable of modulating signal transduction may have, for example, aminoacid deletions and/or insertions and/or substitutions as long as theyretain significant ability to interact with some or all relevantcomponents of a MKK signal transduction pathway. A functional derivativeof MKK may be prepared from a naturally occurring or recombinantlyexpressed MKK by proteolytic cleavage followed by conventionalpurification procedures known to those skilled in the art.Alternatively, the functional derivative may be produced by recombinantDNA technology by expressing parts of MKK which include the functionaldomain in suitable cells. Functional derivatives may also be chemicallysynthesized. Cells expressing MKK may be used as a source of MKK, crudeor purified for testing in these assays.

MKK signal transduction activity may be measured by standard biochemicaltechniques or by monitoring the cellular processes controlled by thesignal. To assess modulation of kinase activity, the test molecule isadded to a reaction mixture containing MKK and a substrate. The kinasereaction is then initiated with the addition of ATP. An immunoassayusing an antiphosphotyrosine antibody is performed on the kinasereaction to detect the presence or absence of the phosphorylatedtyrosine residues on the substrate or to detect phosphorylated tyrosineresidues on autophosphorylated MKK, and results are compared to thoseobtained for controls i.e., reaction mixtures not exposed to the testmolecule.

Uses of MKK Polynucleotide

An MKK polynucleotide may be used for diagnostic and/or therapeuticpurposes. For diagnostic purposes, an MKK polynucleotide may be used todetect MKK gene expression or aberrant MKK gene expression in diseasestates, e.g., acute megakaryocytic leukemia or acute megakaryocyticmyelosis. Included in the scope of the invention are oligonucleotidesequences, that include antisense RNA and DNA molecules and ribozymes,that function to inhibit translation of an MKK. In a specific embodimentof this aspect of the invention, an anti-MKK1 antisense molecule isshown to inhibit MKK-1 protein synthesis resulting in reducedmegakaryocyte growth and differentiation.

Diagnostic Uses of an MKK Polynucleotide

An MKK polynucleotide may have a number of uses for the diagnosis ofdiseases resulting from aberrant expression of MKK. For example, theMKK1 DNA sequence may be used in hybridization assays of biopsies orautopsies to diagnose abnormalities of MKK1 expression; e.g., Southernor Northern analysis, including in situ hybridization assays. Suchtechniques are well known in the art, and are in fact the basis of manycommercially available diagnostic kits.

Therapeutic Uses of an MKK Polynucleotide

An MKK polynucleotide may be useful in the treatment of various abnormalconditions. By introducing gene sequences into cells, gene therapy canbe used to treat conditions in which the cells do not proliferate ordifferentiate normally due to underexpression of normal MKK orexpression of abnormal/inactive MKK. In some instances, thepolynucleotide encoding an MKK is intended to replace or act in theplace of a functionally deficient endogenous gene. Alternatively,abnormal conditions characterized by overproliferation can be treatedusing the gene therapy techniques described below.

Abnormal proliferation of megakaryocytis is an important component of avariety of disease states such as acute megakaryocytic leukemia,myelofibrosis, or acute megakaryocytic myelosis. Recombinant genetherapyvectors, such as viral vectors, may be engineered to express variant,signalling incompetent forms of MKK which may be used to inhibit theactivity of the naturally occurring endogenous MKK. A signallingincompetent form may be, for example, a truncated form of the proteinthat is lacking all or part of its catalytic domain. Such a truncatedform may participate in normal binding to a substrate but lack enzymaticactivity. Thus recombinant gene therapy vectors may be usedtherapeutically for treatment of diseases resulting from aberrantexpression or activity of an MKK. Accordingly, the invention provides amethod of inhibiting the effects of signal transduction by an endogenousMKK protein in a cell comprising delivering a DNA molecule encoding asignalling incompetent form of the MKK protein to the cell so that thesignalling incompetent MKK protein is produced in the cell and competeswith the endogenous MKK protein for access to molecules in the MKKprotein signalling pathway which activate or are activated by theendogenous MKK protein.

Expression vectors derived from viruses such as retroviruses, vacciniavirus, adeno-associated virus, herpes viruses, or bovine papillomavirus, may be used for delivery of recombinant MKK into the targetedcell population. Methods which are well known to those skilled in theart can be used to construct recombinant viral vectors containing an MKKpolynucleotide sequence. See, for example, the techniques described inManiatis et al., 1989, Molecular Cloning A Laboratory Manual, ColdSpring Harbor Laboratory, N.Y. and Ausubel et al., 1989, CurrentProtocols in Molecular Biology, Greene Publishing Associates and WileyInterscience, N.Y. Alternatively, recombinant MKK molecules can bereconstituted into liposomes for delivery to target cells.

Oligonucleotide sequences, that include anti-sense RNA and DNA moleculesand ribozymes that function to inhibit the translation of an MKK mRNAare within the scope of the invention. Anti-sense RNA and DNA moleculesact to directly block the translation of mRNA by binding to targetedmRNA and preventing protein translation. In regard to antisense DNA,oligodeoxyribonucleotides derived from the translation initiation site,e.g., between −10 and +10 regions of an MKK nucleotide sequence, arepreferred.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA. The mechanism of ribozyme action involves sequencespecific hybridization of the ribozyme molecule to complementary targetRNA, followed by a endonucleolytic cleavage. Within the scope of theinvention are engineered hammerhead motif ribozyme molecules thatspecifically and efficiently catalyze endonucleolytic cleavage of MKK1RNA sequences.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences, GUA, GUU and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for predicted structuralfeatures such as secondary structure that may render the oligonucleotidesequence unsuitable. The suitability of candidate targets may also beevaluated by testing their accessibility to hybridization withcomplementary oligonucleotides, using ribonuclease protection assays.

Both anti-sense RNA and DNA molecules and ribozymes of the invention maybe prepared by any method known in the art for the synthesis of RNAmolecules. These include techniques for chemically synthesizingoligodeoxyribonucleotides well known in the art such as for examplesolid phase phosphoramidite chemical synthesis. Alternatively, RNAmolecules may be generated by in vitro and in vivo transcription of DNAsequences encoding the antisense RNA molecule. Such DNA sequences may beincorporated into a wide variety of vectors which incorporate suitableRNA polymerase promoters such as the T7 or SP6 polymerase promoters.Alternatively, antisense cDNA constructs that synthesize antisense RNAconstitutively or inducibly, depending on the promoter used, can beintroduced stably into cell lines.

Various modifications to the DNA molecules may be introduced as a meansof increasing intracellular stability and half-life. Possiblemodifications include but are not limited to the addition of flankingsequences of ribo- or deoxy-nucleotides to the 5′ and/or 3″ ends of themolecule or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the oligodeoxyribonucleotide backbone.

Methods for introducing polynucleotides into such cells or tissueinclude methods for in vitro introduction of polynucleotides such as theinsertion of naked polynucleotide, i.e., by injection into tissue, theintroduction of an MKK polynucleotide in a cell ex vivo, i.e., for usein autologous cell therapy, the use of a vector such as a virus,retrovirus, phage or plasmic, etc. or techniques such as electroporationwhich may be used in vivo or ex vivo.

EXAMPLES Cloning and Characterization of MKK1

For clarity of discussion, the subsection below describes the isolationand characterization of a cDNA clone encoding the novel tyrosine kinasedesignated MKK1. The MKK2 and MKK3 genes were cloned and characterizedusing the same methods.

cDNA Cloning MKK Expression and MKK Characterization

Confluent plates of K-562 cells (ATCC accession number CCL 243) werelysed by treatment with guanidinium-thiocyanate according to Chirgwin etal. (1979, Biochemistry 18:5294-5299). Total RNA was isolated byCsC1-gradient centrifugation. First-strand cDNA was synthesized from 20μg total RNA with avian myeloblastosis virus (AMV) reverse transcriptase(Boehringer Mannheim).

cDNA was used in a polymerase chain reaction under standard conditions(PCR Technology-Principles and Applications for DNA Amplifications, H.E. Erlich, Ed., Stockton Press, New York 1989). Degenerate pools ofprimers corresponding to the amino acid sequence HRDLAA) residues350-355 of SEQ ID NO: 2) and SDVWSF/Y (SEQ ID NO: 24) were prepared andused for the amplification: 5′ oligo pool              H   R   D   L   A5′ GGAATTCC CAC AGN GAC TTN GCN (SEQ ID NO: 20)              T C   A T C   A  A GCN AG 3′ A   C 3′ oligo pool            F/Y   S   W   V   D 5′ GGAATTCC GAA NGT CCA NAC GTC (SEQ IDNO: 21)             ATG  CA         C  S NGA 3′   CThirty-five PCR cycles were carried out using 8 μg (0.8 μg) of thepooled primers. (Annealing 55° C., 1 min; Extension 72° C., 2 min;Denaturation 94° C., 1 min). The reaction product was subjected topolyacrylamide gel electrophoresis. Fragments of the expected size (˜210bp) were isolated, digested with the restriction enzyme EcoRI, andsubcloned into the pBluskript vector (Stratagene) using standardtechniques (Current Protocols in Molecular Biology, eds. F. M. Ausubelet al., John Wiley & Sons, New York, 1988).

The recombinant plasmids were transformed into the competent E. colistrain designated 298.

The subcloned PCR products were sequenced by the method of Sanger et al.(Proc. Natl. Acad. Sci USA 74, 5463-5467) using Sequenase (United StatesBiochemical, Cleveland, Ohio 44111 USA). Clones designated MKK1, MKK2,and MKK3 were identified as novel TKs.

Full-Length cDNA Cloning

The partial cDNA sequence of the new MKK1 TK, which was identified byPCR, was used to screen a λgt11 library from human fetal brain CDNA(Clontech) (complexity of 1×10¹⁰ recombinant phages). One millionindependent phage clones were plated and transferred to nitrocellulosefilters following standard procedures (Sambrook, H. J., MolecularCloning, Cold Spring Harbor Laboratory Press, USA, 1989). The filterswere hybridized to the EcoRI/EcoRI fragment of clone MKK1, which hadbeen radioactively labeled using 50 μCi [α³²P]ATP and the random-primedDNA labeling kit (Boehringer Mannheim). The longest eDNA insert of ˜3500by was digested with the restriction enzymes EcoRI/Sacd to obtain a 5′end probe of 250 bp. This probe was used to rescreen the human fetalbrain library and several overlapping clones were isolated. Thecomposite of the cDNA clones of MKK1, MKK2 and MKK3 is shown in FIGS.1A-1B (SEQ ID NOS 1-2), 2A-2B (SEQ ID NOS 3-4) and 3A-3B (SEQ ID NOS5-6), respectively. The 1.75 million independent phage clones of a humanplacenta library, λZAP, were plated and screened with the 5′ end probe(EcoRI/SacI) of the clone used above. Subcloning of positivebacteriophages clones into pBluskript vector was done by the in vivoexcision protocol (Stratagene).

The composite cDNA sequence and the predicted amino acid sequence ofMKK1, MKK2 and MKK3 are shown in FIGS. 1A-1B (SEQ ID NOS 1-2), 2A-2B(SEQ ID NOS 3-4) and 3A-3B (SEQ ID NOS 5-6), respectively.

MKK Expression

E.coli expression constructs for MKK1, MKK2 and MKK3 were produced bycloning of the corresponding cDNA fragments into a plasmid expressionvector pTZS2 (Ray et al., PNAS USA 89:(13):5705-5709 (1992)) bysubstitution of recoverin coding sequence with synthetic polylinkerfragment. To provide in-frame connection of the coding sequences toprokaryotic translation initiation site coded by the vector, an NdeIrestriction site overlapping start codon (CATATG) was introduced in allthree MKK cDNAs by site directed mutagenesis. The resulting constructsare designed to drive expression of unfused proteins with authenticamino acid sequences. FIG. 8 shows MKK expression constructs.

RNA Blot Analysis of MKKs

Total RNA was isolated from human megakaryocytes, myeloid cells,B-cells, T-cells, and epithelial cells.

PolyA⁺ RNA was isolated on an oligo (dT) column (Aviv and Leder, 1972,Proc. Natl. Acad. Sci. USA 69, 1408-1412). The poly A+ RNA was isolatedusing RNA stat-60 method (Tel-Test B Inc.) and blotted on anitrocellulose filter using a slot blot apparatus (Schleicher andSchuell). 2 μg of poly A⁺ RNA was loaded per lane. The filter washybridized with a ³²P-labeled EcoRI/EcoRI DNA fragment obtained by PCR.Subsequently, the filter was exposed to x-ray film at −70° C. with anintensifying screen. The results, as shown in FIG. 4, suggest that MKK1and MKK2 are preferentially expressed in megakaryocytes. MKK3 expressioncould not be detected using this technique. FIG. 8 shows MKK expressionconstructs.

EXAMPLE Autophosphorylation of MKK2 and MKK3

FIG. 7 represents Western blot analysis of protein from bacteriaexpressing MKK1, MKK2, or MKK3 using an anti-phosphotyrosine antibody(Hansen et al., Electrophoresis 14:112-126 (1993)). All MKK constructswere cloned into the inducible vector pTZS2, and transformed bacteriawere grown under induced and uninduced conditions as described by Ray,et al., (PNAS USA 89:5705-5709 (1992)). Bacterial pellets from thesecultures were resuspended in sample buffer, containing 2-mercaptoethanoland SDS, and boiled. Proteins were separated by SDS-polyacrylamide gelelectrophoresis. The results of this example indicate that MKK2 and MKK3have kinase activity.

EXAMPLE Production of Anti-MKK Antibodies and Immunoprecipitation of MKK

Antibodies recognizing MKK1 and MKK2 protein were made in rabbits usingstandard procedures. The anti-carboxy terminus MKK1 antibody wasgenerated using the synthetic peptide GQDADGSTSPRSQEP (SEQ ID NO 22).The amino-terminus MKK1 Ab was generated using a GST-fusion proteinscontaining 78 amino acids coded by the Smal to BG12 fragment of the MKK1gene. The anti-carboxy terminus MKK2 Ab was made using a syntheticpeptide corresponding to the sequence QQLLSSIEPLREKDKH (SEQ ID NO 23).

MKK1 and MKK2, cloned into the pBluskript plasmid, were transcribed andtranslated in the presence of ³⁵S-methione using standard methods.Following protein synthesis MKK1 and MKK2 were immunoprecipitated (i.p.)with the appropriate rabbit antibodies (Ab) in the presence of SDS. FIG.5 shows immunoprecipitation of in vitro transcribed and translated MKK1and MKK2 proteins.

EXAMPLE Expression of MKK1 Anti-Sense Sequences

Bone Marrow elements isolated from mice treated with 5-flurourocil 6days prior to harvest were infected with retroviruses containingconstructs expressing MKK1, antisense MKK1 (a truncated 5′ EcoRI-PvuIIfragment cloned in the reverse orientation) or the empty retroviralvector (mock). Following infection, cells were cultured and analyzed forthe level of acetylcholinesterase (AChE) as previously described,measured as optical density at 414 nm (Hill, Exp. Hematology 20:354-360(1992). A higher optical density reading indicates a greater AChE leveland correlates with increased megakaryocyte growth and differentiation.Levels of the marine MKK1 protein were determined by metabolicallylabeling cells with ³⁵S-methionine for 12 hours at the end of theexperimental period. Following labeling, cells were lysed and MKK 1protein was isolated by two cycles of immunoprecipitation usinganti-amino terminus MKK1 antibody. The proteins were resolved bypolyacrylamide gel electrophoresis and visualized by autoradiography.

The retroviral construct used (PSR/MSV-Tkneo) was previously described(Mol. Cell. Biol. 11:1785-1792 (1991)). The MKK1 sense constructrepresents the full length gene lacking the poly-adenylation sequences.The MKK1 antisense construct represents the 5′ fragment EcoRI-PvuIIcloned in the reverse orientation. Both the sense and antisenseconstructs are driven by the retroviral long terminal repeat (LTR).

The results of the experiment, as shown in FIGS. 6A-6B, indicate thatexpression of the MKK1 anti-sense sequences in the cultured bone marrowelements is associated with decreased expression of MKK1 and decreasedlevels of ACHE, an indicator of megakaryocyte growth anddifferentiation.

Various modifications of the invention, in addition to those shown anddescribed herein, will become apparent to those skilled in the art fromthe foregoing description. Such modifications are intended to fallwithin the scope of the appended claims. It is also to be understoodthat all base pair sizes given for nucleotides are approximate and areused for purposes of description.

All references cited herein are hereby incorporated by reference intheir entirety.

1. An isolated antibody or antibody fragment that specifically binds toa megakaryocytic kinase (MKK) protein selected from the group consistingof MKK1, MKK2 and MKK3.
 2. The antibody or antibody fragment of claim 1,wherein said MKK protein is MKK1.
 3. The antibody or antibody fragmentof claim 2, wherein said MKK1 comprises the amino acid sequence depictedin SEQ ID NO:
 2. 4. The antibody or antibody fragment of claim 1,wherein said MKK protein is MKK2.
 5. The antibody or antibody fragmentof claim 4, wherein said MKK2 comprises the amino acid sequence depictedin SEQ ID NO:
 4. 6. The antibody or antibody fragment of claim 1,wherein said MKK protein is MKK3.
 7. The antibody or antibody fragmentof claim 6, wherein said MKK1 comprises the amino acid sequence depictedin SEQ ID NO:
 6. 8. The antibody or antibody fragment of claim 1,wherein said antibody or antibody fragment is monoclonal.
 9. Theantibody or antibody fragment of claim 1, wherein said antibody orantibody fragment is chimeric.
 10. A hybridoma that produces theantibody or antibody fragment of claim 1.